Pasumarthi K B, Doble B W, Kardami E, Cattini P A
Department of Physiology, University of Manitoba, Winnipeg, Canada.
J Mol Cell Cardiol. 1994 Aug;26(8):1045-60. doi: 10.1006/jmcc.1994.1125.
Initiation of translation from alternate codons in the same mRNA results in multiple forms of basic fibroblast growth factor (bFGF). High molecular weight species of bFGF make use of leucine translation initiation sites located upstream of the methionine residue used to produce the 18 kiloDalton (kDa) form. Although the addition of exogenous 18 kDa bFGF is known to stimulate DNA synthesis and proliferation of several cell types including embryonic chicken cardiac myocytes, little is known about the role of high molecular weight forms of bFGF. We modified the rat bFGF cDNA to yield high (22/21.5 kDa) or low (18 kDa) molecular weight species of bFGF. Expression of 22/21.5 kDa or 18 kDa bFGF in transfected embryonic chicken ventricular myocyte cultures was confirmed by protein blotting. Expression of both high and low molecular weight species of bFGF was associated with (i) a three-fold increase in overall thymidine incorporation as well as cardiomyocyte labelling index (fraction of cardiomyocyte nuclei incorporating tritiated thymidine); (ii) a two- to three-fold increase in cell number; (iii) an eight-fold increase in protein synthesis; and (iv) a three-fold decrease in myosin accumulation. Subcellular localization of bFGF in the transfected myocyte cultures was also assessed by immunofluorescence microscopy. Over-expression of cDNAs yielding high molecular weight bFGF resulted in predominantly nuclear bFGF staining. By contrast, both cytoplasmic and nuclear staining were observed following over-expression of 18 kDa bFGF. Over-expression of 22/21.5 kDa bFGF was associated with the formation of multiple DNA-containing "clumps" resembling condensed chromatin in cardiac myocyte nuclei. These DNA "clumps" were not observed in cardiac myocyte cultures over-expressing 18 kDa bFGF. These data indicate that over-expression of high as well as low molecular weight forms of bFGF can stimulate cardiac myocyte proliferative potential and decrease myosin accumulation. However, these forms possess distinct subcellular localizations and can have different biological functions in the nucleus.
在同一信使核糖核酸（mRNA）中从不同密码子起始翻译会产生多种形式的碱性成纤维细胞生长因子（bFGF）。高分子量的bFGF利用位于用于产生18千道尔顿（kDa）形式的甲硫氨酸残基上游的亮氨酸翻译起始位点。尽管已知添加外源性18 kDa bFGF可刺激包括胚胎鸡心肌细胞在内的多种细胞类型的DNA合成和增殖，但对于高分子量形式的bFGF的作用了解甚少。我们对大鼠bFGF互补DNA（cDNA）进行了改造，以产生高分子量（22/21.5 kDa）或低分子量（18 kDa）的bFGF。通过蛋白质印迹法证实了22/21.5 kDa或18 kDa bFGF在转染的胚胎鸡心室肌细胞培养物中的表达。高分子量和低分子量bFGF的表达均与以下情况相关：（i）总的胸苷掺入量以及心肌细胞标记指数（掺入氚标记胸苷的心肌细胞核的比例）增加了三倍；（ii）细胞数量增加了两到三倍；（iii）蛋白质合成增加了八倍；（iv）肌球蛋白积累减少了三倍。还通过免疫荧光显微镜评估了bFGF在转染的肌细胞培养物中的亚细胞定位。产生高分子量bFGF的cDNA的过表达导致主要是细胞核bFGF染色。相比之下，18 kDa bFGF过表达后观察到细胞质和细胞核染色。22/21.5 kDa bFGF的过表达与心肌细胞核中形成多个类似于浓缩染色质的含DNA“团块”有关。在过表达18 kDa bFGF的心肌细胞培养物中未观察到这些DNA“团块”。这些数据表明，高分子量和低分子量形式的bFGF的过表达均可刺激心肌细胞的增殖潜能并减少肌球蛋白积累。然而，这些形式具有不同的亚细胞定位，并且在细胞核中可能具有不同的生物学功能。
